Metallo-organic salt compounds and pharmaceutical uses thereof

ABSTRACT

The present invention provides a complex compound having the structure: ##STR1## wherein R 1  and R 1 , are the same or different and each is an alkyl group, a phenyl group or a substituted derivative of a phenyl group; 
     wherein R 2  and R 2 , are the same or different and each is hydrogen, an unbranched alkyl group, a halide or a group having the structure ##STR2## wherein R is hydrogen, an alkoxide group, an alkyl group, or OH; wherein R 3  and R 3 , are the same or different and each is hydrogen or an alkyl group; 
     wherein X and X&#39; are the same or different and each is a water soluble group having weak to intermediate ligand field strength; and 
     Q is a soluble, pharmaceutically acceptable negative ion. 
     The invention also provides a method of treating a subject having a condition associated with the presence of free radicals in quantities sufficient to cause undesirable symptoms. A method of treating a wound or a burn and a method for treating a subject afflicted with tumor cells so as to cause regression of the tumor cells are also provided. These methods involve administering to the subject (or topically administering to the burn or wound) an effective amount of the compound. 
     Furthermore, the invention provides pharmaceutical compositions for the treatment of tumor cells in a subject or for the treating of undesirable symptoms associated with the presence of free radicals. The compositions comprising an effective amount of the compound and a pharmaceutically acceptable carrier. Also provided is an antimicrobial composition which comprises a suitable carrier and the compound in an amount effective to suppress the growth of microorganisms.

This is a continuation of application Ser. No. 279,417, filed Dec. 2, 1988 now U.S. Pat. No. 5,049,557, which, in turn, is a continuation-in-part of application Ser. No. 147,713, and application Ser. No. 147,714, both filed Jan. 25, 1988, now U.S. Pat. Nos. 4,866,054 and 4,866,053, which, in turn are continuation-in-parts of application Ser. No. 862,804, filed May 13, 19886, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to metallo-organic cobalt compounds and their use in the treatment of subjects for inflammatory conditions, particularly arthritis, and like conditions caused by oxygen free radicals. The invention also relates to a method of treating burns and wounds and the use of the subject compounds as antimicrobial agents.

It has been recognized for some time that inflammation in mammalian species can be traced at least in part to active oxygen species, including superoxide, and radicals associated therewith at the inflammatory site. Considerable research has been undertaken to measure and detect oxygen radicals, to establish the mechanisms whereby enzymes such as superoxide dismutase are effective in countering oxygen radical toxicity, and in the development and use of copper amine oxidases in preventing tissue damage and even in promoting damagetissue recovery. Recently superoxide dismutas, which decomposes highly toxic oxygen free radicals, has been put into veterinary use as an anti-inflammatory agent with efficacy in the treatment of conditions such as traumatic arthritis of horses.

Free radical toxicity has also been identified as operative in poisonings by various pharmacologic agents. Enzymes such as superoxide dismutase have been used as antidotes to nullify the toxic effects of the putative free radical generators in vivo.

However, the compounds which have been developed heretofore for active-oxygen or superoxide antagonism and destruction in vivo have not proven as effective as desired, or are characterized by side reactions, or cannot be made in commercially significant quantities at reasonable cost.

The present invention provides compounds and methods for the treatment of subjects having inflammatory conditions or other conditions associated with free radicals whereby the above-mentioned drawbacks are eliminated. The invention provides a method for treating a subject having a condition resulting from active oxygen or superoxide toxicity which may result in acute or chronic inflammation or other disorders, such as arthritis.

The invention also provides a method of treating burns and wounds in a subject. The treating of burns of the first, second, and third degree has long been and remains one of the most difficult medical problems. The criteria for success of any method for treating a burn includes proper contraction of the wound, epithelialization, hair follicle preservation, and the assessment of newly formed granulation tissue. Contraction represents the difference between the initial wound size of the burn and the size of the burn twelve days later (12th post burn day or PBD), which includes both open and healed areas calculated as a percentage of the initial wound size.

Epithelialization represents the percentage of the newly covered areas of the burn surface on the 12th PBD out of the total wound area on that same day. The presence of hair follicles indicates maintenance or restoration of dermal microcirculation and prevention of tissue ischemia and thus ischemic and postischemic damage. The preservation of hair follicles and their count should be carried out microscopically in tissue sections. Also important in the evaluation of medicament of treating burns is the assessment of newly formed granulation tissue. The thickness of the new collagen layer synthesized in the healing burn should be measured on PBD 12.

As part of the overall management of burn wounds, a topically antibacterial agent, such as silver sulfadiazine, may be applied. Unexpectedly, it has been found that the compounds of the present invention may be used as an antibacterial agent which can help prevent the colonization of the wound by pathologic agents. The method of the present invention maximizes epithelialization of the burn on a macroscopic level and maximizes hair follicle preservation on a microscopic level.

Additionally, it has been unexpectedly found that the compounds of the present invention cause regression in the growth of tumor cells in vivo. A method is provided for the treatment of a subject afflicted with a tumor which comprises administering to the subject the compounds of the present invention in an amount sufficient to cause regression of the tumor cells.

SUMMARY OF THE INVENTION

The present invention provides a complex compound having the structure: ##STR3## wherein R₁ and R₁, are the same or different and each is an alkyl group, a phenyl group or a substituted derivative of a phenyl group;

wherein R₂ and R₂, are the same or different and each is hydrogen, an unbranched alkyl group, a halide or a group having the structure ##STR4## wherein R is hydrogen, an alkoxide qroup, an alkyl group, or OH; wherein R₃ and R₃, are the same or different and each is hydrogen or an alkyl group; and

wherein X and X' are the same or different and each is a water soluble group having weak to intermediate ligand field strength.

The invention also provides a complex compound having the structure: ##STR5## wherein R₁ and R₁, are the same or different and each is an alkyl group, a phenyl group or a substituted derivative of a phenyl qroup;

wherein R₂ and R₂, are the same or different and each is an unbranched alkyl group, a halide or a group having the structure ##STR6## wherein R is hydrogen, an alkoxide group, an alkyl group, or OH; wherein R₃ and R₃, are the same or different and each is hydrogen or an alkyl group;

wherein X and X' are the same or different and each is a water soluble group having weak to intermediate ligand field strength; and

Q is a soluble, pharmaceutically acceptable negative ion.

The invention also provides a method of treating a subject having a condition associated with the presence of free radicals in quantities sufficient to cause undesirable symptoms. A method of treating a wound or a burn and a method for treating a subject afflicted with tumor cells so as to cause regression of the tumor cells are also provided. The methods of the present invention involve administering to the subject (or topically administering to the burn or wound) a compound having the structure: ##STR7## wherein R₁ and R₁, are the same or different and each is an alkyl group, a phenyl group or a substituted derivative of a phenyl group;

wherein R₂ and R₂, are the same or different and each is hydrogen, an unbranched alkyl group, a halide or a group having the structure ##STR8## wherein R is hydrogen, an alkoxide group, an alkyl group, or OH; wherein R₃ and R₃, are the same or different and each is hydrogen or an alkyl group;

wherein X and X' are the same or different and each is a water soluble group having weak to intermediate ligand field strength; and

Q⁻ is a soluble, pharmaceutically acceptable negative ion.

Furthermore the invention provides pharmaceutical compositions for the treatment of tumor cells in a subject or for the treating of undesirable symptoms associated with the presence of free radicals. The compositions comprise an effective amount of the compound and a pharmaceutically acceptable carrier. Also provided is an antimicrobial composition which comprises a suitable carrier and the compound in an amount effective to suppress the growth of microorganisms.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B: A Diagram of the Molecular Orbitals for the Cobalt Complex.

FIG. 2(A) and 3(A): Graph of Body Weight vs. Days for Antiascites Activity of Compound 23.

FIG. 2(B) and 3(B): Graph of % Viability vs. Days for Antiascites Activity of Compound 23.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a complex compound having the structure: ##STR9## wherein R₁ and R₁, are the same or different and each is an alkyl group, a phenyl group or a substituted derivative of a phenyl group;

wherein R₂ and R₂, are the same or different and each is hydrogen, an unbranched alkyl group, a halide or a group having the structure ##STR10## wherein R is hydrogen, an alkoxide group, an alkyl group, or OH; wherein R₃ and R₃, are the same or different and each is hydrogen or an alkyl group; and

wherein X and X' are the same or different and each is a water soluble qroup having weak to intermediate ligand field strength.

Also provided is a complex compound having the structure: ##STR11## wherein R₁ and R₁, are the same or different and each is an alkyl group, a phenyl group or a substituted derivative of a phenyl group;

wherein R₂ and R₂, are the same or different and each is an unbranched alkyl- qroup, a halide or a group having the structure ##STR12## wherein R is hydrogen, an alkoxide group, an alkyl group, or OH; wherein R₃ and R₃, are the same or different and each is hydrogen or alkyl group;

wherein X and X' are the same or different and each is a water soluble group having weak to intermediate ligand field strength; and

Q is a soluble, pharmaceutically acceptable negative ion.

The compounds of the present invention may be crystallized with numerous counteranions. Those which are pharmaceutically acceptable and are water soluble, such as halide ions, PF₆ ⁻ and BF₄ ⁻, are preferred. The bromine salts of the present compounds are the most preferred because they are the easiest to crystallize and are more water soluble than other salts of the compounds.

As discussed above the R₁ and R₁, groups of the compound may be the same or different from each other and each may be an alkyl group, a phenyl group or a substituted derivative of a phenyl group. Preferably the alkyl group is a C₁ -C₅ group with methyl, ethyl and butyl groups being particularly preferred. Suitable substituted derivatives of the phenyl group are derivatives wherein each substituent is a halide, an alkyl group or a group having the structure ##STR13## where R is hydrogen, an alkoxide group, an alkyl group or an OH group. To date the most useful derivatives have proven to be those in which the substituents are halides, carbonyl groups or alkyl groups.

The R₂ and R₂, groups of the complex compounds of the present invention may also be the same or different and may be hydrogen, an unbranched alkyl group, a halide or a group having the structure ##STR14## where R is hydrogen, an alkoxide qroup, an alkyl group or an OH group. In certain embodiments, it is preferred that R₂ and R₂, are chlorine or hydrogen atoms or a C₁ -C₃ alkyl group. In embodiments where R₂ has a structure ##STR15## it is preferred that R is hydrogen, a methyl group or an OH group.

The R₃ and R₃, of the complex compounds of the present invention are the same or different and each may be hydrogen or an alkyl group, preferably a C₁ -C₃ alkyl group.

With respect to the X and X' groups, it is preferred that these groups are water-soluble and have a weak to intermediate ligand field strength. Ligands are arranged in a spectrochemical series according to the magnitude of their field strength or the "Δ₀ " they bring about. The symbol Δ₀ represents the difference between the energies of the d_(xy), d_(xz), and d_(yz) -orbitals and the d_(z) ² and d_(x) ² _(-y) ² orbitals. From experimental studies, it is known that the order of ligands, based on their ligand field strength, is approximately the same for the complexes of all the transition metals in their common oxidation states with only an occasional inversion of order between ligands that stand near to one another. A typical order of some common ligands is as follows:

    I.sup.- <Br.sup.- <Cl.sup.- <F.sup.- <OH.sup.- <C.sub.2 O.sup.2- <H.sub.2 O<NH.sub.3 <en<NO.sub.2 .sup.- <CN.sup.-

The cyanide ion, which stands at the opposite end of the series from the halide ions, has the strongest ligand field strength and induces the largest d-orbital splitting of any ligand listed. On the other hand, the halide ions such as Br⁻ and Cl⁻ have weak ligand field strength and induces the smallest d-orbital splitting. Ligands such as NH₃ and OH⁻ have intermediate ligand field strength. For general background information, see F. Albert Cotton and Geoffrey Wilkinson, "Advanced Inorganic Chemistry", John Wiley & Sons, 4th ed., p. 663. In the present invention, it is preferred that X and X' are ligands with weak to intermediate ligand field strength, such as halides or NH₃, H₂ O, or dimethyl sulfoxide.

Particular preferred embodiments of the complex compounds of the present invention are as follows: ##STR16## wherein Ph is a phenyl group.

The invention also provides a pharmaceutical composition and method for treating a subject having a condition associated with the presence of free radicals in quantities sufficient to cause undesirable symptoms. The pharmaceutical composition comprises a pharmaceutically acceptable carrier and the compound of the present invention in an amount effective to alleviate the undesirable symptoms associated with the presence of the free radicals. The method comprises administering to the subject the compound in an amount effective to alleviate the undesirable symptoms.

The method may be used for the treatment of any condition associated with free radicals. It is however most effective against conditions associated with oxygen free radicals, such as O₂ ⁻. Such conditions may comprise inflammation, including synovial inflammation and arthritis, ulcers such as diabetic ulcers, and conditions caused by the loss of circulation due to free radicals, such as hair loss due to the loss of dermal microcirculation.

Preferably, the method is used in the treatment of acute or chronic arthritis with a dosage of the compounds of 0.1 to 250 mg/kg (especially about 1 to about 200 mg/kg) of body weight, but in all cases at most 50% of the LD₅₀ value of the compound, when the compound is administered orally as is preferred. However, the complex compound or a combination of the complexes can be administered subcutaneously or even topically in a suitable vehicle, e.g. physiological saline in the case of s.c. administration and dimethylsulfoxide (DMSO) in the case of a topical administration, although ointments, salves or like conventional vehicles may be employed.

For oral administration, the complex or mixture of complexes may be prepared in suitable dosage forms. For example it may be prepared as dragees, as capsules, as tablets, as an elixir or other oral dosage form.

The dose may be administered one to six times daily, depending upon the severity of the inflammatory condition, preferably under medical supervision so that the dosage can be reduced or the number of daily administrations limited as the inflammatory condition subsides.

The compounds of the invention may also have prophylactic properties in preventing the spread of arthritic inflammation and have been found to be effective in reducing the severity of the actual condition which develops in subjects who are prone to such inflammatory states. The compounds may also be effective in preventing postischemic heart damage and for geriatric applications other than antiarthritics.

It is also contemplated that the compounds of the invention may be used in conjunction with known antiinflammatory agents with propionic acid side chains, especially indomethacine, to further alleviate the suffering of arthritic inflammation.

The invention also provides an antimicrobial composition which comprises a suitable carrier and the compound of the present invention in an amount effective to suppress the growth of microorganisms. The antimicrobial composition or compounds of the present invention may be used for the treatment of a wound or burn by topically administering to the wound or burn the composition or compounds. The compounds may also be used in the treatment of conditions which are normally treated with antimicrobial or antibacterial agents. For example, the compounds may be used in the topical treatment of an infectious disease or an abrasion as an antibiotic.

The cobalt complexes of the present invention are water-soluble and may be dissolved in a number of carriers. Suitable carriers include polar, protic solvents such as water or especially normal saline. The cobalt complexes may also be suspended in a suspension medium that is not miscible with water, for example petrolatum.

The concentration of the complexes in the solvent or suspension medium can vary from 0.1 to 50 mg/ml. A preferred concentration range lies between 1 and 10 mg/ml.

The complexes may be applied to the site of the burn, wound, abrasion, etc. in the form of an aerosol, in the form of a salve, ointment, or cream, or directly in a liquid solvent, preferably normal saline, by the use of a medicine dropper. Furthermore the complexes may be applied to the burn site together with a topical anaesthetic agent such as benzocaine, a soothing agent such as menthol, an antibacterial agent such as bacitracin, or a combination of these ingredients.

The method is particularly effective for killing microorganisms such as Strep. β hemolytic, Strep. α hemolytic, Enterococci, Staph. coagulase (+), Staph. coagulase (-), E. Coli, Klebsiella, Pseudomonas, Proteus, or C. albicans.

It has also been found that the compounds of the present invention are effective in the treatment of subjects having tumors. Administration of the compounds to a subject afflicted with tumor cells causes a regression in the growth of the tumor cells. The compound may be directly administered to the subject or it may be administered in a pharmaceutical composition which comprises an effective anti-tumor amount of the compound and a pharmaceutically acceptable carrier. This method has been found to be particularly effective in the treatment of tumors associated with ascites cells.

It is also contemplated that the compounds and compositions of the present invention may be used in the treatment of other conditions associated with free radicals, such as poisonings with pharmacologic agents or conditions caused by ionizing radiation. The compounds may also be used in industrial applications where free radicals, such as oxygen radicals, are undesirable; for example, in wastewater treatment as an oxygen scavenger.

Other embodiments and uses for the compounds of the present invention will become apparent to those skilled in the art upon a reading of the present disclosure. These uses and embodiments are intended to be within the spirit and scope of the present invention.

The invention is further illustrated in the Experimental Details section which follows. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter.

EXPERIMENT DETAILS I. Synthesis

Several cobalt(III) complex compounds having the general formula [CoL(NH₃)₂ ]⁺, wherein L represents a tetradentate ligand with two oxygen and two nitrogen donor atoms, have been prepared and characterized. The purity of the compounds was established by elemental analysis, N.M.R. and U.V.-visible spectroscopy. The geometry around the cobalt atom was established by X-ray crystallography. Analytical reagent grade chemicals were used without further purification. A detailed description for a typical preparation for each of the compounds follows. Of the compounds described only [Co(L23)(NH₃)₂ ]⁺ have been reported previously.sup.(1).

1. Synthesis of [CoL23(NH₃)₂ ]Cl, (L23=N,N'-bis(acetylacetone)ethylenediimine) ##STR17##

The tetradentate ligand was prepared as described by McCarthy.sup.(2). To 0.023 moles of L23 dissolved in 100 ml of methanol was added 0.016 moles of CoCl₂ H₂ O dissolved in 100 ml of methanol. Concentrated NH₄ OH was added dropwise with continuous stirring until the pH became basic (pH=8). Stirring was continued for an additional three hours. The resulting precipitate was filtered and recrystallized from hot ethanol/water.

Found; C, 40.9%; H. 6.75%; N, 16.1%. Calculated for C₁₂ H₂₄ O₂ N₄ CoCl; C, 41.09%; H, 6.90%; N, 15.9%. MW=350.4.

2. Synthesis of [Co(L64)(NH₃)₂ ]Cl, (L64=N,N'-bis(benzoylacetoneethylenediimine). ##STR18##

L64 was prepared as described by McCarthy.sup.(2). To 0.022 moles of L64 dissolved in 100 ml of CH₂ Cl₂ was added a filtered solution of 0.020 moles of CoCl₂ 6H₂ O in 100 ml of absolute methanol. A concentrated solution of NH₄ OH was added slowly with continuous stirring until the solution became basic (pH=8). Stirring continued for an additional 4 hrs. The solution was concentrated to a volume of 50 ml and filtered. Slow evaporation of the mother liquor gave the desired product.

Calculated for C₂₂ H₂₈ O₂ N₄ CoCl.H₂ O: C, 54.72%; H, 6:20%; N, 11.61%; Found: C, 54.61%; H, 6.04%; N, 11.49%. MW=482.4

3. Synthesis of [Co(L67)(NH₃)₂ ]Cl, (L67=N,N'-bis(chloroacetylacetone)ethylenediimine) ##STR19##

L67 was prepared as previously described.sup.(3). [Co(L67)(NH₃)₂ ]Cl was prepared by the same procedure as [CoL23)(NH₃)₂ ]Cl. Found; C, 34.61%; H, 5.38%; N, 13.03%. Calculated for C₁₂ H₂₂ O₂ N₄ CoCl₃ ; C, 34.35%; H, 5.02%; N, 13.35%. MW=419.32

4. Synthesis of [Co(L68)(NH₃)₂ ]Br, (L68=N,N'-bis(chlorobenzoylacetone)ethylenediimine) ##STR20##

L68 was prepared by direct chlorination of L64 with a slight excess of N-chlorosuccinimide (NClS).sup.(4). 0.1 moles of L64 was dissolved in 700 ml of ice cold CH₂ CL₂. 0.25 moles of NClS was added and the reaction stirred for 20 min. The ligand precipitated as a yellow powder which was filtered, washed with ether and dried. 0.005 moles of L68 was suspended in 100 ml of absolute ethanol under nitrogen to which 0.01 moles of KOH dissolved in 25 ml of methanol was added. 0.0045 moles of Co(OAc)₂.4H₂ O in 25 ml of methanol was slowly added to the above suspension with continuous stirring. The reaction mixture was heated to 50° C. for 1 hr. After cooling, the orange precipitate of [Co(L68)] was filtered, washed with ethanol and dried. 0.002 moles of the Co(II) complex [CoL68] was suspended in 100 ml of methanol under N₂. 1 ml of 30% H₂ O₂ was added dropwise and anhydrous ammonia was bubbled through the reaction mixture until all the Co(II) complex had been dissolved. At this point, bubbling of N₂ was discontinued and the solution was stirred for an additional 1 hr. The solution was filtered and three equivalents of NaBr dissolved in a minimum amount of water was added. Slow evaporation of the solution yielded the desired product.

Found: C, 43.44%; H, 4.46%; N, 9.07%. Calculated for C₂₂ H₂₆ BrCl₂ N₄ O₂ Co.H₂ O: C, 43.58%; H, 4.65%; N, 9.24%. MW=605.78

5. Synthesis of [Co(L69)(NH₃)₂ ]Cl (L69=N.N'-bis(methylacetylacetone)ethylenediimine) ##STR21##

L69 was prepared by condensation of anhydrous ethylenediamine with methylacetylacetone.sup.(5). The complex is prepared by the same procedure as described for [Co(L23)(NH₃)₂ ]Cl.

Calculated for C₁₄ H₂₈ N₄ O₂ CoCl.H₂ O; C, 42.42%, H, 7.57, N, 14.14%. Found: C, 42.14%; H, 7.48%; N, 14.01%. MW=396.4

6. Synthesis of [Co(L70)(NH₃)₂ ]Br, (L70=N,N'-bis(methylbenzoylacetone)ethylenediimine) ##STR22##

L70 was prepared by condensation of ethylene diamine with methylbenzoylacetone.sup.(6). The Co(III) complex was prepared via the Co(II) complex by the procedure described for [Co(L68)NH3)2]⁺.

Found: C, 46.87%; H, 5.03%; N, 8.95%. Calculated for C₂₄ H₃₂ B₁.N₄ O₂ Co:47.07%; H, 5.27%; N, 9.15%, MW=611.9.

All complexes described can be crystallized with other counteranions. Those anions which give the best solubility in water were chosen here.

The reaction of Co(II) complexes with molecular oxygen has been studied extensively.sup.(7,8). Normally, cobalt(II) forms 2:1 peroxo bridged complexes in aqueous solutions.sup.(8). In recent years, a number of Co(II) complexes have been reported to give 1:1 cobalt-oxygen adducts at room temperature. These complexes usually contain ligands which when bound to Co(II) give rise to a low spin planar geometry. Addition of base and O₂ to these complexes leads to the formation of octahedral complexes where the base and the O₂ occupy axial positions.sup.(9).

On the basis of measurements utilizing a variety of physical techniques it is now a well accepted fact that the most accurate electronic structure description for the Co:O₂ moiety is a Co(III) ion bound to O₂ ⁻, where the actual amount of Co→O₂ electron transfer depends on the nature of the ligand and the donor set.sup.(9,10). It has been shown that electron transfer increases with increase of the ligand field strength.sup.(7). This can be easily understood from the molecular orbital diagram depicted in FIG. 1.

In FIG. 1, the donor atoms A define the basal plane of the molecule, while B represents the axial ligands. As the ligand field strength of the donor atoms around the cobalt increases, the metal orbitals (especially d_(z) ² and d² -y² which are α orbitals) are raised in energy relative to the π orbitals of O₂ and more electronic charge is transfered from the metal to the bound dioxygen, i.e., the O₂ molecule attains more O₂ ⁻ character. Thus, considering electronic structural arguments only, one can conclude that a Co(III) complex with a set of six donor atoms (four A donors, and two B donors) having an intermediate ligand field strength could be effective in reacting with O₂ ⁻ by either forming a stable Co(III)-O₂ adduct (the O₂ ⁻ substitutes one of the B ligands), or by oxidizing O₂ ¹, liberating dioxygen and yielding a Co(II) complex. The relative strength of the different ligands is well known from the spectrochemical series. However, it should be quite clear that a sterically unstrained ligand system which can easily accomodate both metal oxidation states [Co(II), Co(III)] with minimal reorqanization of the geometry around the metal and which does not lead to a high spin Co(II) complex should facilitate the reaction.

Thus, on the basis of geometrical, steric and electronic requirements we suggest that a Co(III) complex having a quadridentate ligand which imposes planarity on the octahendral basal plane should be a suitable candidate for reaction with the O₂ ⁻ radical anion. The effectiveness of this reaction will depend on the nature of the quadridentate ligand, its ligand field strength and on the nature of the axial ligands B.

The complexes [CoL(NH₃)₂ ]⁺ fit nicely with the set of requirements given above. First, the six donor atoms N₄ O₂ give rise to an intermediate ligand field. Secondly, the quadridentate ligand L when bound to Co(III) gives rise to a 6,5,6 ring system where the six membered rings are unsaturated, thereby ensuring the planarity of the complex basal plane without steric strain, as has been determined from X-ray crystal structure analysis. Thirdly, the ligand L when bound to Co(II), gives a low spin, planar complex.

It should be pointed out that the unsaturation of the six-membered rings is important not only because it ensures the required geometry, but also because it provides for an effective pathway for transmitting electronic effects of different substituents to the cobalt center, thereby affecting the relative energies of the metal d orbitals.

II. In vivo Inflammatory Studies

The drugs used are prepared just before performing the experiment. Drugs are dissolved at a concentration of 10⁻² M (or 2×10⁻² M or 4×10⁻² M) in pyroqen free sterile saline. The dissolved drug is then filtered through a sterile and pyrogen-free 0.2 micron filter (Acrodisc, Gelman).

Procedure

8-12 CD-1 female mice (Charles River), age 2-5- months are numbered, weighed and distributed to 2-3 cages, 3-4 mice in each cage. 0.2 ml of pyroqen-free saline or drug are injected subcutaneously in a randomized order.

Thirty minutes after injecting the drugs or the saline, the right paw of each mouse is injected with 25 microliters of 1% carrageenin (viscarin type, Marine Colloids) in pyrogen-free saline or with 5 microliters (0.227 U) of xanthine oxidase (Sigma).

1.5 hr to 2 hrs after injecting the inflammatory stimulus to the right paw, both paws of the animal are amputated at the knee joint and weighed. The uninjected left paw serves as an internal control for the degree of swelling of the right paw in each animal.

In some experiments the surface temperature of the right and left paws were recorded also before the amputation was performed.

Calculations

The difference in mg between the weight of the right and left paw in control animals (injected with saline) represents 100% of the acute inflammatory response. Concomitantly, the difference between paws of the drug treated animals is calculated and compared to control. Results for [CoL23(NH₃)₂ ]Cl (designated as "23" in tables) are shown in Tables 1, 2 and 3 which follows.

                  TABLE 1                                                          ______________________________________                                         Effect of several products on carrageenin paw                                  oedema in mice measured by paw weight.                                                                Paw Weight                                                     No. of  total   route of                                                                              average                                                                               %     % inhi-                             Product                                                                               expts.  mice    admin. mg/kg  activity                                                                             bition-                             ______________________________________                                         saline --      --      *S.C.  --      100% 0                                   23     4       14      *S.C.  25     63.1% 36.9%                               ______________________________________                                    

                  TABLE 2                                                          ______________________________________                                         Effect of several products on xanthine-oxidase                                 paw oedema measured by temperature reduction                                          No. of  total   route of                                                                              average                                                                               %     % inhi-                             Product                                                                               expts.  mice    admin. mg/kg  activity                                                                             bition-                             ______________________________________                                         saline --      --      *S.C.  --     100%  0                                   23     2       6       *S.C.  22.7    31%  69%                                 ______________________________________                                    

                  TABLE 3                                                          ______________________________________                                         LD.sub.50 and ED.sub.50 of various drugs on xanthine                           oxidase and carrageenin paw oedema in mice                                                          :xanthine                                                 stimulus             oxidase  carrageenin                                      measurement          :temp.   weight mg                                        Drug    LD.sub.50    ED.sub.50                                                                               ED.sub.50                                        mg/kg                mg/kg    mg/kg                                            ______________________________________                                         23      75           <38      25                                               ______________________________________                                    

Compound 64 ([Co(L64)(NH₃)₂ ]Cl) was found to be as effective as or more effective than compound 23 in corresponding tests. Both compounds were found to be effective in reducing oedema upon oral administration in force feeding with pills containing 8 mg of active ingredient and administered in a quantity sufficient to provide an effective dose. The pills were enterally coated (see below).

EXAMPLE 1

The composition of tablets is as follows:

    ______________________________________                                         active ingredient (one or both                                                                         25.0   mg.                                             of compounds 23 or 64                                                          corn starch             97.0   mg.                                             polyvinyl pyrrolidone   175.0  mg.                                             magnesium stearate      3.0    mg.                                                                     300.0  mg.                                             ______________________________________                                    

The active ingredient and the corn starch are wetted by an aqueous polyvinyl pyrrolidone solution of approx. 15% w/v, followed by granulation, and drying of the wet granules at about 40°-45° C. The dried granulate is thoroughly mixed with magnesium stearate, and the mixture so obtained is further processed by a tablet machine, equipped with an appropriate pressing tool, to give tablets of 300 mg. weight containing 25 mg. of active ingredient. One manufacturing lot includes 100 tablets.

EXAMPLE 2

Dragees of the following composition are prepared:

    ______________________________________                                         active ingredient (one or both                                                                         50.0   mg.                                             of compounds 23 or 64)                                                         lactose                 94.0   mg.                                             polyvinyl pyrrolidone   4.0    mg.                                             magnesium stearate      2.0    mg.                                                                     150.0  mg.                                             ______________________________________                                    

Granulates are prepared according to Example 1, and from them dragee kernels of 150 mg. weight are pressed. The dragee kernels are coated with a layer containing sugar and talc followed by coloring with an approved food colorant and polishing with beeswax.

EXAMPLE 3

25 mg. of active ingredient (one or both of compounds 23 or 64) are dissolved in 100 ml. of distilled water. The solution is filled into 500 ampules. In this way ampules containing 2 ml. of a solution containing 25 mg./ml. of active agent each are obtained. The contents of an ampule are injected subcutaneously.

EXAMPLE 4

Gelatin capsules of the following composition are prepared:

    ______________________________________                                         active ingredient (one or both                                                                         25.0   mg.                                             of compounds 23 or 64)                                                         maize starch            122.0  mg.                                             colloidal silica        3.0    mg.                                                                     150.0  mg.                                             ______________________________________                                    

The ingredients are homogenized, and the put into hard gelatine capsules. 1000 capsules of 150 mg. (filling) weight each, containing 25.0 mg. of active ingredient per capsule, make a lot.

EXAMPLE 5

Pills of the active ingredient are enterally coated with a melt of 45 parts of n-butyl stearate, 30 parts of carnauba wax and 25 parts of stearic acid, all by weight, at a temperature of 75° C.

II. Superoxide Scavenqinq

Compounds 67, 68, and 69 ([Co(L67)(NH₃)₂ ]Cl;[Co(L68)(NH₃)₂ ]Br; [Co(L69)(NH₃)₂ ]Cl, respectively) were tested spectrophotometrically for O₂ quenching (reduction of NBT in the presence of xanthine and xanthine oxidase as the generating system of superoxide radicals). These compounds were also evaluated by quenching of O₂ produced by macrophages in tissue culture. Macrophages are a source of superoxide radicals in inflammatory states.

The activity of these compounds were assessed and compared to compounds 23 and 64 which hereinabove are reported to be effective superoxide radical scavengers and effective in the treatment of rat adjuvant arthritis. Subsequently, these two compounds were also found to be effective in burn wound healing. Table 4 summarizes the results obtained with compounds 67, 68, 69 as compared with 23 and 64.

As depicted in Table 4 compounds 23 and 67 have given similar results both in the test tube and with macrophages.

50% O₂ ⁻ scavenging was achieved at a concentration of about 10⁻⁵ M whereas compounds 64 and 68 gave diverse results with the two methods: in the test tube the compounds were very effective, yielding 50% O₂ ⁻ quenching at concentrations of 2-8×10⁻⁶ M whereas in the macrophage system there was a decrease in superoxide scavenging ability to the range of 10⁻⁴ M. Compound 69 was not as effective in O₂ scavenging as judged by either method. BSA was used as a chelating agent, in order to determine the stability of each ligand with the metal in physiological fluids. Compound efficacy in the presence of BSA was as follows: 23>67>69>64.,

                                      TABLE 4                                      __________________________________________________________________________     The drug concentration [M] at which 50% superoxide quenching is                achieved,                                                                      as measured by spectrophotometric and tissue culture methods. Also, the        concentration of the drug at which 50% of protein synthesis is                 inhibited in cultured macrophages.                                                                             50% inhibition of                              Compound                                                                             Test tube Macrophages     protein synthesis                              #     determination(a)                                                                         -BSA(b)   +BSA(c)                                                                              (d)                                            __________________________________________________________________________     23    7.7 ± 0.5 × 10.sup.-5                                                           7.0 ± 0.8 × 10.sup.-5                                                           1 × 10.sup.-4                                                                  6.0 × 10.sup.-5                          64    8.0 ± 0.64 × 10.sup.-6                                                          1.4 ± 0.23 × 10.sup.-4                                                          6 × 10.sup.-4                                                                  6.4 × 10.sup.-5                          67    7.4 ± 0.9 × 10.sup.-5                                                           5.5 ± 0.5 × 10.sup.-5                                                           2 × 10.sup.-4                                                                  6.5 × 10.sup.-5                          68    2.6 ± 0.2 × 10.sup.-6                                                           2.0 ± 0.2 × 10.sup.-4                                                           not tested                                                                           1.0 × 10.sup.-4                          69    5.0 ± 0.04 × 10.sup.-4                                                          3.7 ± 0.7 × 10.sup.-4                                                           3 × 10.sup.-4                                                                  6.0 × 10.sup.-5                          __________________________________________________________________________      Table 4 gives the comparative activity of each compound as determined by       various methods of measuring superoxide scavenging. The values represent       the concentration of each compound at which 50% superoxide inhibition was      achieved ± SEM. These are the averages of 3 experiments each done in        duplicate.                                                                     (a) xanthinexanthine oxidase system.                                           (b) and (c) macrophage system, without or in the presence of .15% BSA as       chelating agent, respectively.                                                 (d) Drug concentration at which 50% of protein synthesis in the cells is       inhibited.                                                               

III. Anti-ascites (tumor) activity of compound 23

The effect of compound 23 on ascites tumor development in C57/B1 mice was tested. Each mouse was injected intraperitoneally with either 5×10⁵ or 1×10⁶ Ehrlich ascites cells. Half of the mice received injections or 12-16 mg/kg of compound 23 and half remained untreated. Body weight and survival were tested at the time intervals indicated in FIGS. 2 and 3. The results of two (out of seven) representative experiments are depicted in FIGS. 2 and 3. The main conclusions so far are: a) the compound inhibits tumor proliferation and delays mortality caused by the tumor very considerably; b) experiments with doses (toxicity) and intervals between injections are needed; and c) the results are encouraging because the amount of tumor cells injected is very high and kills 100% of the animals within 20-21 days.

IV. Burn/wound healing for Compounds 23 and 67

Female and male Hartley derived albino guinea-pigs were used throughout this study. They were housed individually and fed ad libitum normal guinea-pig's chow and water, supplemented with 1 gm of Vitamin C per week.

The back of each animal was clipped and depilated 24 hours prior to the burn injury. Two symmetrical mirror-image round burns were inflicted on the back of each animal with an aluminum template heated to 75° C. and applied for 5 seconds under general anaesthesia (ketamine 150 mg/kg I.M.). Intraortic india ink injection indicated in our preliminary studies that the injuries were deep second-degree burns. The burn areas were dressed in the same fashion as is routinely done in cases of such burns.

Application of the compounds for Experiments 3, 4 and 5 was carried out as follows: five times a day for the first four days and twice daily from the fifth to the 12th day. On day twelve the animals were sacrificed and the wound tissues were sent for histological examination.

Control wounds were treated with 1 mg bovine serum albumin/1 ml saline or with 1 ml of saline only. Catalase treatment was also applied in 1 mg/ml saline. Compound 23 was applied at either 3 mg/ml or varying concentrations of 3 mg/ml for the first four days, 2 mg/ml twice daily from the 5th to the 8th day and 1 mg/ml twice daily from the 9th to the 12th day.

The wounds were selected for treatment at random. Three or four treatment groups were performed in each experiment. All dressings were changed under general anaesthesia every four days, at which time wound tracing for healing analysis was performed.

The evaluation of healing is based upon the following four criteria:

1. Epithelialization--wound epithelialization expressed as percent was calculated employing the following formula: ##EQU1## where E₁ =rate of epithelialization expressed in percent.

A12total=wound size on the 12th day post burn (both opened a healed wound).

Ao=area of open wound on the same 12th day.

E1 represents the % of the newly covered area out of the total wound area on the 12th day. E2 represents the % of the healed area on the 12th day out of the total area of the primary wound induced at zero time.

2. Contraction--The burn wound size as delineated by its outer boundaries was traced on a transparent polyethylene sheet on post burn days (PBD) 4, 8 and 12. Areas were measured by a system composed of an IBM PC computerized video-camera interfaced with specially designed software. The percentage of contraction was calculated according to the following formula ##EQU2## where: C--percentage of contraction on PBD 12.

A₁ --initial burn wound size.

A₁₂ --burn wound size PBD 12 (opened and healed wound) Excessive contraction leads to varying imitation of use of healed areas degrees of limitation of use of healed areas and is thus unfavorable.

3. Assessment of the newly formed granulation tissue--Since the evaluation of collagen synthesis is precluded in burn wound models, the newly formed granulation tissue in each burn wound was assessed histologically on PBD 12. Four adjacent sections were taken from the center of each wound. Employing the Mason's Trichrome staining method aided to delineate the newly formed granulation tissue which originated from the non-burned dermal layer in each section.

4. Hair follicle count--The preservation of hair follicles and their count was quantified. The presence of hair follicles and their regeneration indicated preservation of blood circulation and the degree of healing of the underlying tissues of the dermis.

Results

1. Epithelialization--Tables 5 and 6--is clearly superior in the wounds receiving Compound 23 treatment being 50.1% to 81.4% for various Compound 23 concentrations, as compared with the average of 36.2% of the control wound (34.4% and 38.6% accordingly) which were treated with either BSA or saline only. Catalase treatment prevented new epithelial formation almost completely--and most of the wound remained unhealed.

2. Contraction--is a natural process occurring in each healing wound and too much contraction during healing may cause organ dysfunction. The results in Tables 5 and 6 indicate that Compound 23, particularly in the higher concentration, did not enhance contraction and may actually somewhat reduce this process.

3. Granulation tissue formation--In the five control wounds and three Compound 23 treated wounds analyzed from experiment no. 3, the thickness of the newly formed collagen in controls was 539 on the average whereas in Compound 23 treated wounds it was 467. This constitutes 86% of the collagen formed in untreated healing indicating that Compound 23 does not cause and any reduce fibrosis. This means that the compound reduces the amount of scar tissue and allows more normal tissue to be formed in the treated wounds.

4. Hair Follicle Count--The presence of hair follicles indicates maintenance of dermal microcirculation and prevention of tissue ischemia and thus ischemic and postischemic damage. The preservation of hair follicles and their count were undertaken; the number of regenerated hair follicles were counted microscopically in tissue sections. The biological section was projected onto a video, colored screen, connected to a color camera. Hair follicles were counted in each wound, in three to eight microscopic fields, in each of 4-8 histological sections. Each field was 2 mm long and 5 microns thick. The mean and standard error (SE) were calculated for each wound and for the whole treatment group. See especially Tables 8, 9 and 10 which appear hereinafter.

                  TABLE 5                                                          ______________________________________                                         experiment no. 3 (10 animals - 20 wounds)                                                Epithelialization-%                                                           E1       E2       Contraction-%                                       ______________________________________                                         control    34.4       21.3     35.8                                            Compound 23                                                                               50.1       31.7     35.7                                            3 mg/ml                                                                        catalase    6.1        2.8     41.4                                            ______________________________________                                    

                  TABLE 6                                                          ______________________________________                                         experiment no. 4 (11 animals - 22 wounds)                                                Epithelialization-%                                                           E1       E2       Contraction-%                                       ______________________________________                                         control    38.6       22.5     40.1                                            Compound 23                                                                               81.4       51.6     37.5                                            3 mg/ml                                                                        Compound 23                                                                               61.3       33.6     42.1                                            3/2/1 mg                                                                       ______________________________________                                    

In the subsequent experiment no. 5, we used 20 guinea pigs and inflicted 32 wounds. 14 control wounds were treated with saline. 9 wounds were treated with Compound-64 (3.8 mg/ml). 9 wounds were treated with Compound-23 mg/ml.

Table 7 summarizes the results of this experiment, with respect to % epithelialization and % contraction.

                  TABLE 7                                                          ______________________________________                                         experiment no. 5                                                                       % Epithelialization = E1                                                                       % Contraction                                          ______________________________________                                         control   69.2*             44.4                                               Compound 64                                                                              97.3**            42.1                                               3.8 mg                                                                         Compound 23                                                                              91.3**            37.0                                               ______________________________________                                          *higher control values over previous experiments due to changed frequency      of application of dressings.                                                   **virtually total recovery                                               

With respect to epithelialization the two compounds 23 and 64 show considerable improvement as compared to untreated. Contraction was significantly changed by the treatments.

The following Tables 8, 9 and 10 each summarizes all the parameters which were assessed for experiments #3, #4, #5 respectively.

There was no substantially significant difference in the initial wound size among the treatment groups of each experiment as should be expected.

Contraction was significantly different amongst the various treatment groups in experiments #3 and #4. However, in experiment #5 contraction was significantly lower in the Compound 23 treated group as compared to controls (Table 7).

Contraction is a natural process occurring in each healing wound. Excessive contraction may lead to varying degrees of organ limitation and is thus unfavorable. Contraction with the various treatments given and especially with compound 23 did not exceed control values and was actually reduced in experiment #5. This indicates that the drug does not heal by producing excessive fibrosis. A fibrotic scar is less aesthetic and limits the functionality of the healed area.

In experiment #3 (Table 8) epithelialization was only 6% in catalase treated group, 34.4% for control-BSA treated wound and 50% for compound 23. Epithelialization with compound 23 treatment was superior to control treated wounds, it was not statistically significant. Thus catalase treatment was significantly worse than either the control or compound 23 treatments.

In experiment #4 (Table 9) treatment with 3 mg of compound 23 significantly improved epithelialization to 89% as compared with 38.5% in control treated wounds. The use of decreasing amounts of compound 23 (treatment C) also improved epithelialization, but to a lesser degree.

In experiment #5 (Table 10) the wound dressings were not changed on the 4th day, but only on the 8th and 12th PBD. The reduced dressing in experiment #5 resulted in improved healing of the wounds of all groups compared with experiments #3 and #4. Epithelialization with 3 mg of compound 23 was 90.3%, meaning that wounds were almost completely healed on day 12. Another Co compound was introduced, compound 64, and it also dramatically improved epithelialization to 97%.

Across the three experiments reported hereinabove, epithelializaiton was superior in all the groups treated with Co compounds. This should be considered in light of the crucial role of epithelialization in the process of healing.

Hair follicle preservation and formation is crucial in the assessment of wound healing since it also indicates the maintenance of dermal microcirculation and prevention of tissue ischemia.

Hair follicle preservation in experiment #3 (Table 8) was very significantly better with compound 23 than either catalase or control treated groups, the amount of hairs per field microscopic was twice as much in the compound 23 treated group; 15.5 in compound 23 vs. 8.2 and 9.2 in control and catalase treatments, respectively.

Experiment #4 (Table 9) exhibited the same phenomenon, 3 mg of compound 23 and decreasing amounts of compound 23 were superior to the control group, though treatment with a higher compound 23 concentration was superior to decreasing amounts of the same treatment, i.e. 15.4 and 13.1 hair follicles per field of compound 23 treated groups respectively vs 6.9 hair follicles for control. Very surprisingly compound 64, which proved to be an inducer of epithelialization, did not improve hair follicle preservation over control values, and was significantly lower than treatment with compound 23.

Across the three experiments reported hereinabove compound 23 proved superior with respect to hair follicle preservation which coincided with superior epithelialization, and this represents healing on a macroscopic level. Hair follicle preservation was twofold higher than control or any other treatment suggesting that microcirculation injury and ischemia resulting from superoxide radical production were at least partially prevented by the use of compound 23 as superoxide radical scavenger.

The thickness of the newly formed granulation tissue on PBD 12 was not fully assessed but in both experiments 3 and 4 (Tables 8, 9) the collagen formed in the compound 23 treated group was slightly thicker (10% to 15%) than control values difference which was not statistically significant. The fact that the layer of new collagen did not exceed that of control by more than 10% to 15% taken together with the fact that contraction was lower due to treatment indicates that a more aesthetic scar will result from our treatment with compound 23.

The data indicate that burn wound healing in this quinea pig model was significantly accelerated in the groups treated especially with Co-compounds, i.e. compounds 23 and 64. This improvement was accompanied by a significant increase in epithelialization. Moreover, compound 23 also demonstrated improved hair follicle preservation, a fact which indicates that the microcirculation in the burn area was at least partially protected and therefore regenerated better during the healing process. The fact that contraction and new granulation tissue did not increase by compound 23 indicates that the scar tissue will not become excessively fibrotic and thus will result in a more aesthetic scar and a more functional healing organ.

                                      TABLE 8                                      __________________________________________________________________________     Assessment of burn wound healing by means of                                   contraction, epithelialization, hair follicle                                  preservation, and newly formed granulation tissue.                             exp. 3                                                                         Female guinea pigs = 10, wounds (n) = 20                                       Weight = 530 + 10 gr.                                                          Treatments:                                                                           A (n = 6) catalase                                                                          1 mg/ml/treatment                                                 B (n = 7) BSA = control                                                                     1 mg/ml/treatment                                                 C (n = 7) Co-BAE-23                                                                         3 mg/ml/treatment                                                   Treatment                                                                      A         B        C                                                           Catalase  BBA = control                                                                           Co-BAE-23                                          __________________________________________________________________________     Initial wound                                                                           1249.13 ± 25.64                                                                       1283.4 ± 68.7                                                                         1267 ± 37.94                                   size - mm.sup.2                                                                Contraction                                                                             41.47 ± 4.73                                                                          35.83 ± 4.04                                                                         35.71 ± 2.42                                    PBD-12(%)                                                                      Epithelialization                                                                       6.05 ± 6.05                                                                            34.4 ± 11.50                                                                         50.06 ± 12.14*                                 PBD-12(%)                                                                      Hair follicles                                                                          9.25 ± 3.3                                                                            8.18 ± 2.15                                                                           15.5 ± 1.38**                                  per 2 mm × 5                                                             Hair follicles                                                                          113.4     100      189**                                              % of control                                                                   New collagen -                                                                          not       654.6 ± 16.0                                                                         730.72 ± 44.6                                            assessed                                                              __________________________________________________________________________      *statistically significant difference from control                             **very significant difference from control                               

                                      TABLE 9                                      __________________________________________________________________________     Assessment of burn wound healing, by means of                                  contraction, epithelialization, hair follicle                                  preservation, and newly formed granulation tissue.                             exp. 4                                                                         Male animals = 11, wounds (n) = 22                                             Weight = 602.7 ± 24 gr.                                                     Treatments:                                                                    A (n = 8) Co-BAE-23                                                                             3 mg/ml/treatment                                             B (n = 7) control-saline                                                                        1 ml/treatment                                                C (n = 7) Co-BAE-23                                                                             3 mg/ml/treatment 1-4 PBD                                                      2 mg/ml/treatment 5-8 PBD                                                      1 mg/ml/treatment 9-12 PBD                                              Treatment                                                                      A        B        C                                                            Co-BAE-23                                                                               Saline   Co-BAE-23                                                    3 mg     control  3/2/1 mg                                           __________________________________________________________________________     Initial wound                                                                            1466.23 ± 77.08                                                                      1435.74 ± 53.19                                                                      1566.13 ± 105                                   size - mm.sup.2                                                                Contraction                                                                              36.14 ± 2.89                                                                         40.08 ± 1.88                                                                         42.13 ± 2.94                                    PBD-12(%)                                                                      Epithelialization                                                                         89.25 ± 5.38**                                                                      38.55 ± 11.60                                                                         52.56 ± 16.21*                                 PBD-12(%)                                                                      Hair follicles                                                                            15.4 ± 0.9**                                                                         6.9 ± 2.06                                                                           13.1 ± 2.72*                                   per 2 mm × 5                                                             New collagen -μM                                                                      616.6 ± 46                                                                           576.5 ± 22.3                                                                         675.3 ± 79                                      __________________________________________________________________________      *some statistical significance from control                                    **very significant difference from control                               

                                      TABLE 10                                     __________________________________________________________________________     Assessment of burn wound healing, by means of                                  contraction, epithelialization, hair follicle                                  preservation, and newly formed granulation tissue.                             exp. 5                                                                         Female animals = 20, wounds (n) = 32                                           Weight = 532.5 + 6.02 gr.                                                      Treatments:                                                                           A (n = 9) Co-BAE-64                                                                         3.0 mg/ml/treatment                                               B (n = 14) control-saline                                                                   1 ml/treatment                                                    C (n = 9) Co-BAE-23                                                                         3 mg/ml/treatment                                          __________________________________________________________________________              Treatment                                                                      A        B       C                                                             BBAE-64  Saline control                                                                         Co-BAE-23                                            __________________________________________________________________________     Initial wound                                                                           1384.76 ± 35.7                                                                       1398 ± 34.1                                                                         1294.0 ± 33.9                                     size - mm.sup.2                                                                Contraction                                                                             42.03 ± 1.89                                                                         44.89 ± 3.01                                                                         38.4 ± 3.17**                                    PBD-12(%)                                                                      Epithelialization                                                                        97.29 ± 1.51**                                                                      69.25 ± 4.5                                                                          90.3 ± 5.33**                                    PBD-12(%)                                                                      Hair follicles                                                                            9.9 ± 0.83**                                                                       10.6 ± 1.25                                                                          15.3 ± 2.39**                                    per 2 mm × 5                                                             Hair follicles                                                                          93.4     100     144**                                                % of control                                                                   New collagen -                                                                          not      not     not                                                           assessed assessed                                                                               assessed                                             __________________________________________________________________________              Treatment                                                                      A        B       C                                                             C-23     C-67    Saline                                               __________________________________________________________________________     Initial wound                                                                           1171.84 ± 132                                                                        1117.6 ± 58.1                                                                       1107.37 ± 125.23                                  size - mm.sup.2                                                                Contraction                                                                             29.16 ± 11.09                                                                        26.23 ± 9.42                                                                        29.07 ± 6.8                                       PDN-16%                                                                        Epithelialization                                                                       57.5 ± 29.4                                                                          69.17 ± 12                                                                             57.4 ± 18.98*(a)                               PBD-16(%) (E1)                                                                 Epithelialization                                                                       38.98 ± 31.74                                                                        50.81 ± 11.21                                                                          41.8 ± 12.05*(b)                               PBD-16(5) (E2)                                                                 __________________________________________________________________________      Epithelialization (E1)  represents the % of the newly covered area of the      wound surface on the 16th post burn day out of the total wound area on th      same day.                                                                      Epithelialization (E2)  represents the % of the newly covered area of the      wound surface on the 16th post burn day out of the initial wound area on       the first day.                                                                 *marginally statistically significant from control.                            **very significant difference                                            

The following examples are directed to the preparation of pharmaceutical composition for the typical treatment of burns.

EXAMPLE I

An aerosol composition is prepared having the following proportions:

    ______________________________________                                         Benxocaine       1.00%                                                         Camphor          0.10                                                          Menthol          0.10                                                          Pyrilamine Maleate                                                                              0.25                                                          Bacitracin       0.02                                                          Acetulan.sup.R   1.00                                                          (Acetylated landin                                                             alcohols)                                                                      Oleyl Alcohol    4.00                                                          Dipropylene Glycol                                                                              1.00                                                          Compound 23      0.3                                                           Propellant 152 a/II                                                                             92.23                                                                          100.00%                                                       ______________________________________                                    

EXAMPLE II

The following water-soluble ointment is prepared having the following proportions:

    ______________________________________                                         Polyethylene Glycol 200                                                        ______________________________________                                         Monostearate       15.0%                                                       Veegum             5.0                                                         Polysorbate 80     1.0                                                         Methylparaben      0.1                                                         Compound 23        0.3                                                         Purified water     78.6                                                                           100.00%                                                     ______________________________________                                    

EXAMPLE III

The following oleaginous ointment is prepared having the following proportions:

    ______________________________________                                         Compound 64        .38%                                                        Petrolatum balance to                                                                            100%                                                         ______________________________________                                    

V. Evaluation of the potential of compound 23 in the presence of human serum Material and Methods

The microorganisms, growth conditions, MIC and MBC tests were performed in the same conditions as in the first parts. But instead of using BH and TSB as growth media, sterile human serum was used. The microorganisms were incubated in human serum with different concentrations of compound 23, for establishing the MIC, while the MBC was determined on TSA as before.

Results and Discussion

The results summarized in Table 12 indicate that:

1) The gram (-) microoganisms show the same pattern as if they were grown in the rich medium (BH), the MBC is 4 mg/ml approximately.

2) The gram (+) microorganisms grown in the serum are as sensitive as those grown in BH except for Staph. Coagulase positive which is less sensitive in serum (MBC+3.2 mg/ml). The reason may be that this microorganism is able to coagulate the serum and thereby influence the mode of action of compound 23.

                                      TABLE 12                                     __________________________________________________________________________     Sensitivity of gram positive and gram                                          negative bacteria to C.sub.23 compound grown in human serum.                   __________________________________________________________________________             Microorganism                                                                  Strep. β                                                                       Strep. α                                                                              Staph. Staph.                                                hemolytic                                                                           hemolytic                                                                            Enterococci                                                                           coagulase (+)                                                                         coagulase (-)                                 C.sub.23 (mg/ml)                                                                       MBC* MBC   MBC    MBC    MBC                                           __________________________________________________________________________     0.4     5.10.sup.4                                                                          3.10.sup.4                                                                           4.10.sup.3                                                                            5.10.sup.4                                                                            1.10.sup.4                                    0.8     1.10.sup.4                                                                          5.10.sup.2                                                                           4.10.sup.2                                                                            1.10.sup.4                                                                            1.10.sup.3                                    1.6     8.10.sup.3                                                                          1.10.sup.2                                                                           2.10.sup.2                                                                            1.10.sup.3                                                                            5.10.sup.2                                    3.2     5.10.sup.3                                                                          5.10.sup.1                                                                           2.10.sup.1                                                                            1.10.sup.2                                                                            2.10.sup.2                                    6.4     1.10.sup.2                                                                          1.10.sup.1                                                                           1.10.sup.1                                                                            1.10.sup.1                                                                            1.10.sup.1                                    __________________________________________________________________________             E. Coli                                                                             Klebsiella                                                                           Pseudomonas                                                                           Proteus                                                                               C. albicans                                           MBC  MBC   MBC    MBC    MBC                                           __________________________________________________________________________     2       8.10.sup.3                                                                          8.10.sup.3                                                                           8.10.sup.3                                                                            3.10.sup.3                                                                            3.10.sup.3                                    4       8.10.sup.2                                                                          2.10.sup.2                                                                           7.10.sup.3                                                                            4.10.sup.2                                                                            1.10.sup.3                                    8       6.10.sup.1                                                                          3.10.sup.1                                                                           1.10.sup.3                                                                            8.10.sup.1                                                                            1.10.sup.2                                    16      0  .sup.                                                                            0  .sup.                                                                             1.10.sup.2                                                                            1.10.sup. 1                                                                           1.10.sup.2                                    __________________________________________________________________________      *MBC = Minimal Bactericidal Concentration.                                     The initial concentration of microorganisms was 5.10.sup.4 /ml.          

VI. Comparison of the sensitivity of ten different strains of clinical isolates of Psedomonas and the strain of Staph. coagulase positive to compound 23

The purpose of these experiments was to compare ten strains of Pseudomonas and Staph. (+) which showed various patterns of sensitivity to antibiotics, to their sensitivity to compound 23 compositions.

Material and Methods

Microorganisms--Ten clinical isolates of Pseudomonas aeruginosa and ten of Staph. (+) with various patterns of sensitivity to antibiotics were tested.

Growth conditions--As mentioned is the first part, but were tested only on TBS medium.

MIC and MBC--as described above.

Results and Discussion

Although many of the strains tested (Pseudomonas as well as Staph. coagulase positive) were very resistant to many antibiotics tested (as Gentamicin and all the new cephalosporins), their sensitivity to compound 23 compositions was very similar (Table 13). Compound 23 for Pseudomonas was bactericidal at 4 mg/ml, whereas for Staph. (+) it was bactericidal 1.5 mg/ml-3.0 mg/ml.

                                      TABLE 13                                     __________________________________________________________________________     Sensitivity to C-23 of various strains of clinical isolates.                   __________________________________________________________________________     (a) Pseudomonas                                                                Microorganism                                                                  Ps.(1)        Ps.(2) Ps.(3) Ps.(4) Ps.(5)                                      C.sub.23 mg/ml                                                                       MIC.sup.1                                                                          MBC.sup.2                                                                          MIC                                                                               MBC MIC                                                                               MBC MIC                                                                               MBC MIC                                                                               MBC                                      __________________________________________________________________________     2     -   4.10.sup.4                                                                         -  5.10.sup.4                                                                         -  4.10.sup.4                                                                         -  3.10.sup.4                                                                         -  5.10.sup.4                               4     -   1.10.sup.3                                                                         -  4.10.sup.4                                                                         -  1.10.sup.2                                                                         -  1.10.sup.3                                                                         -  1.10.sup.3                               8     -   2.10.sup.2                                                                         -  1.10.sup.1                                                                         -  0  .sup.                                                                           -  0  .sup.                                                                           -  0  .sup.                                 16    -   0  .sup.                                                                           -  1.10.sup.1                                                                         -  0  .sup.                                                                           -  0  .sup.                                                                           -  0  .sup.                                 __________________________________________________________________________     Ps.(6)        Ps.(7) Ps.(8) Ps.(9) Ps.(10)                                           MIC MBC MIC                                                                               MBC MIC                                                                               MBC MIC                                                                               MBC MIC                                                                               MBC                                      __________________________________________________________________________     2     -   1.10.sup.4                                                                         -  5.10.sup.4                                                                         -  2.10.sup.4                                                                         +  1.10.sup.4                                                                         +  2.10.sup.4                               4     -   0  .sup.                                                                           -  2.10.sup.3                                                                         -  6.10.sup.3                                                                         -  0  .sup.                                                                           -  8.10.sup.1                               8     -   0  .sup.                                                                           -  0  .sup.                                                                           -  0  .sup.                                                                           -  0  .sup.                                                                           -  0  .sup.                                 16    -   0  .sup.                                                                           -  0  .sup.                                                                           -  0  .sup.                                                                           -  0  .sup.                                                                           -  0  .sup.                                 __________________________________________________________________________     (b) Staph. coagulase positive                                                  Staph (+)                                                                      1             2      3      4      5                                                 MIC MBC MIC                                                                               MBC MIC                                                                               MBC MIC                                                                               MBC MIC                                                                               MBC                                      __________________________________________________________________________     0.2   +       +      +      +      +                                           0.4   +       +      +      +      +                                           0.75  +       -  8.10.sup.3                                                                         +      +      -  1.10.sup.4                               1.5   -   1.10.sup.4                                                                         -  0  .sup.                                                                           -  4.10.sup.4                                                                         -  4.10.sup.1                                                                         -  6.10.sup.1                               3     -   0  .sup.                                                                           -  0  .sup.                                                                           -  6.10.sup.2                                                                         -  0  .sup.                                                                           -  0  .sup.                                 __________________________________________________________________________     6             7      8      9      10                                                MIC MBC MIC                                                                               MBC MIC                                                                               MBC MIC                                                                               MBC MIC                                                                               MBC                                      __________________________________________________________________________     0.2   +       -  3.10.sup.4                                                                         -  2.10.sup.4                                                                         -  8.10.sup.4                                                                         -  5.10.sup.4                               0.4   +       -  1.10.sup.4                                                                         -  8.10.sup.3                                                                         -  1.10.sup.4                                                                         -  9.10.sup.3                               0.75  -   2.10.sup.4                                                                         -  1.10.sup.4                                                                         -  6.10.sup.3                                                                         -  1.10.sup.4                                                                         -  9.10.sup.3                               1.5   -   8.10.sup.3                                                                         -  8.10.sup.3                                                                         -  0   .sup.                                                                          -  1.10.sup.4                                                                         -  3.10.sup.3                               3     -   1.10.sup.2                                                                         -  1.10.sup.2                                                                         -  0  .sup.                                                                              8.10.sup.3                                                                         -  1.10.sup.2                               __________________________________________________________________________      1. MIC = Minimal Inhibitory Concentration. (+) visible growth; (-)             transparent.                                                                   2. MBC = Minimal Bactericidal Concentration.                                   The initial concentration of microorganisms was 5.10.sup.4 /ml.          

Sensitivity of various microorganisms were tested to:

(a) Silver sulfadiazine (SSD)

(b) Sulfamylon (SSD)

(c) Compound 23.

The purpose of this study was to compare the sensitivity of SSD and SM, which are well known antimicrobial agents, to compound 23.

The microorganisms, growth conditions, MIC and MBC tests are performed as described in the first part, on TSB medium.

Results and Discussion

Silver Sulfadiazine is effective against the strains tested (gram positive and negative as well) at low concentration (bactericidal at 50 μ/ml) in comparison with compound 23 (Table 14).

Sulfamylon is effective at low concentrations (75-150 μg/ml) only for the Streptococci, whereas for the other gram (+) tested (namely, Staphylococci or Enterococci) or gram (-) very high concentrations are needed to be used to get a bactericidal effect (12,000 μg/ml). This concentration is much higher than that of compound 23. C. albicans is not sensitive even at that concentration.

                                      TABLE 14                                     __________________________________________________________________________     Sensitivity of gram positive and gram negative                                 bacteria to Silver Sulfa Diazine (SSD) and Sulfamylon (SM).                    __________________________________________________________________________     Microorganism                                                                  SSD Strep. β                                                                          Strep. α                                                                         Enterococci                                                                            Staph (+)                                                                              Staph (-)                                  μg/ml                                                                           MIC.sup.1                                                                          MBC.sup.2                                                                          MIC MBC MIC MBC MIC MBC MIC MBC                                    __________________________________________________________________________        25                                                                              -   7.10.sup.4                                                                         -   7.10.sup.4                                                                         +       +       +                                             50                                                                              -   7.10.sup.3                                                                         -   8.10.sup.1                                                                         +       -   7.10.sup.3                                                                         -   5.10.sup.4                                75                                                                              -   0  .sup.                                                                           -   0  .sup.                                                                           -   7.10.sup.4                                                                         -   1.10.sup.3                                                                         -   1.10.sup.3                               100                                                                            150                                                                          __________________________________________________________________________             SSD E. Coli Klebsiella                                                                             Pseudomonas                                                                            Proteus                                            μg/ml                                                                           MIC MBC MIC MBC MIC MBC MIC MBC                                    __________________________________________________________________________                25                                                                             50                                                                              -   8.10.sup.3                                                                         -   1.10.sup.3                                                                         -   5.10.sup.4                                                                         -   0  .sup.                                          75                                                                              -   2.10.sup.2                                                                         -   0  .sup.                                                                           -   3.10.sup.3                                                                         -   0  .sup.                                         100                                                                              -   0  .sup.                                                                           -   0  .sup.                                                                           -   3.10.sup.1                                                                         -   0  .sup.                                         150   0  .sup.                                                                               0  .sup.                                                                               0  .sup.                                                                               0  .sup.                               __________________________________________________________________________     Microorganism                                                                  SM  Strep. β                                                                          Strep. α                                                                         Enterococci                                                                            Staph (+)                                                                              Staph (-)                                  μg/ml                                                                           MIC.sup.1                                                                          MBC.sup.2                                                                          MIC MBC MIC MBC MIC MBC MIC MBC                                    __________________________________________________________________________        75                                                                              -   2.10.sup.3                                                                         -                                                                    150                                                                              -   9.10.sup.2                                                                         -   1.10.sup.3                                                       300                                                                              -   6.10.sup.2                                                                         -   1.10.sup.3                                                       600                                                                              -   6.10.sup.2                                                                         -   1.10.sup.3                                                        3000             +   1.10.sup.4                                                                         -   1.10.sup.4                                                                         -   1.10.sup.4                               6000              -   1.10.sup.4                                                                         -   1.10.sup.4                                                                         -   1.10.sup.4                             12,000              -   5.10.sup.3                                                                         -   5.10.sup.3                                                                         -   3.10.sup.3                             __________________________________________________________________________             SM  E. Coli Klebsiella                                                                             Pseudomonas                                                                            Proteus                                            μg/ml                                                                           MIC MBC MIC MBC MIC MBC MIC MBC                                    __________________________________________________________________________                75                                                                            150                                                                            300                                                                            600                                                                            3000                                                                             +       +       +       +                                                    6000                                                                             -       -       -       +                                                  12,000                                                                             -   1.10.sup.4                                                                         -   1.10.sup.4                                                                         -   1.10.sup.4                                                                         -   1.10.sup.4                             __________________________________________________________________________      1. MIC = Minimal Inhibitory Concentration. (+) visible growth; (-)             transparent.                                                                   2. MBC = Minimal Bactericidal Concentration.                                   The initial concentration of microorganism was 5.10.sup.4 /ml.           

VII. Mutagenicity of compounds #23 and 64

Test was carried out on Photobacterium fisherii as described in Methods in Enzymology Vol. 133:264-284(1986) by S. Ulizur who also carried out the present tests. This test is more sensitive than the Ames test (see reference).

    ______________________________________                                         Conc. in                                                                              #23              #64                                                    mg/ml  growth   luminescence                                                                               growth luminescence                                ______________________________________                                         10     -        0           -      0                                           5      -        0           -      0                                           2.5    -        0           -      0                                           1.25   -        0           -      0                                           0.6    -        0           -      0                                           0.3    -        0           -      0                                           0.15   -        0           -      7                                           0.075  -        0.5         +      130                                         0.037  ++       500         ++     300                                         (control)                                                                             ++       800         ++     800                                         ______________________________________                                    

Conclusion: These compounds are efficient antimicrobials to this gram-negative bacterium. No mutagenic activity is revealed by back mutation to luminescent liminescent state.

The values are given as luminescence (quanta/sec⁻¹ /ml⁻¹). Significant genotoxic effect of a compound is considered when the maximal luminescence developed due to the chemical in question is 3-4 times higher than that obtained with the control.

VIII. Activity of Compounds Outside Defined Class

The following compounds were found to be unreactive, meaning they show no scavenging activity below concentrations of about 10⁻² M: Co(en)₃ Cl₃.3H₂ O (where en=ethylenediamine); Co(pn)₃ Cl₃ (where pn=propylenediamine); [Co(Tim-6,13-(OH)₂)Br₂ ]Br (the compound is shown below and is also toxic); ##STR23##

Perrocene and Ferrocene derivatives (very effective as O₂ ⁻ scavengers but has absolutely no biological activity. In burn wounds, these compounds are worse than the controls). 

What is claimed is:
 1. A complex compound having the structure: ##STR24## wherein R₁ and R₁, are the same or different and each is an alkyl group, a phenyl group or a substituted derivative of a phenyl group;wherein R₂ and R₂, are the same or different and each is hydrogen, an unbranched alkyl group, a halide or a group having the structure ##STR25## wherein R is hydrogen, an alkoxide group, an alkyl group, or OH; wherein R₃ and R₃, are the same or different and each is hydrogen or an alkyl group; and wherein X and X' are the same or different and each is a water soluble group having weak to intermediate ligand field strength.
 2. A complex compound having the structure: ##STR26## wherein R₁ and R₁, are the same or different and each is an alkyl group, a phenyl group or a substituted derivative of a phenyl group;wherein R₂ and R₂, are the same or different and each is an unbranched alkyl group, a halide or a group having the structure ##STR27## wherein R is hydrogen, an alkoxide group, an alkyl group, or OH; wherein R₃ and R₃, are the same or different and each is hydrogen or alkyl group; wherein X and X' are the same or different and each is a water soluble group having weak to intermediate ligand field strength; and Q is soluble, pharmaceutically acceptable negative ion.
 3. A compound of claim 2, wherein Q⁻ is a halide ion, PF₆ ⁻, or BF₄ ⁻.
 4. A compound of claim 3, wherein Q⁻ is Cl⁻ or Br⁻.
 5. A compound of claim 1 or 2, wherein each of R₁ and R₁, is an C₁ -C₅ alkyl group, a phenyl group, or a substituted derivative of a phenyl group where each substituent is a halide, an alkyl group, or a group having the structure ##STR28## wherein R is hydrogen, an alkoxide group, an alkyl group or OH.
 6. A compound of claim 5, wherein each of R₁ and R₁, is a C₁ -C₃ alkyl group.
 7. A compound of claim 5, wherein each of R₁ and R₁, is a substituted derivative of a phenyl group where each substituent is a halide, carbonyl group, or an alkyl group.
 8. A compound of claim 1 or 2, wherein each of R₂ and R₂, is a halide or a group having the structure ##STR29## wherein R is CH₃ or OH.
 9. A compound of claim 8, wherein each of R₂ and R₂, is chlorine.
 10. A compound of claim 1, wherein each of R₂ and R₂, is hydrogen.
 11. A compound of claim 1 or 2, wherein each of R₂ and R₂, is a C₁ -C₃ alkyl group.
 12. A compound of claim 1 or 2, wherein each of R₃ and R₃, is a C₁ -C₅ alkyl group.
 13. A compound of claim 2, wherein each of R₃ and R₃, is a C₁ -C₃ alkyl group.
 14. The compound of claim 1 or 2, wherein each of X and X' is NH₃, H₂ O, a halide, or dimethyl sulfoxide.
 15. A compound of claim 1 having the structure: ##STR30##
 16. A compound of claim 1 having the structure: ##STR31## wherein Ph is a phenyl group.
 17. A compound of claim 2 having the structure: ##STR32##
 18. A compound of claim 2 having the structure: ##STR33## wherein Ph is a phenyl group.
 19. A compound of claim 2 having the structure: ##STR34##
 20. A compound of claim 3 having the structure: ##STR35## wherein Ph is a phenyl group.
 21. A compound of claim 17, 18, 19 or 20, wherein Q⁻ is Br⁻. 